As the human genome project nears completion, the focus of research is shifting to the immense tasks of identifying the structures, functions, and interactions of proteins produced by individual genes, and determining their roles in cancers and other diseases (Marte B. Nature 2003, 422 (Suppl.): 191-237; Liotta L et al., Nat. Reviews 2000, 1:40-56; Emmert-Buck et al., Am J Pathol. 2000, 156:1109-1115). Analysis of tissue proteins and mRNA transcripts is limited by the current technologies for preserving clinical specimens. Traditional formalin-fixed paraffin-embedded (FFPE) specimens provide superior morphology and easy long-term storage of clinical specimens. However, FFPE specimens are not always compatible with current molecular techniques due to suboptimal recovery of most macromolecules.
Because of these extraction problems, frozen tissue is preferred for molecular research. However, large scale processing and storage of frozen tissue are impractical and expensive. In the routine practice of pathology, the need for superior morphology provided by FFPE outweighs the need for molecular diagnosis. This situation becomes even more problematic with limited biopsies. Therefore, a method that could efficiently extract high quality proteins and nucleic acids in sufficient quantities to perform any number of molecular diagnostic methods while providing optimal morphology from FFPE tissue would provide the ideal solution to many of these problems.
All currently available molecule extraction methods require the homogenization or destruction of tissues, fixed or fresh, such that multiple specimens must be prepared for both molecular analysis and histological diagnosis (Clark et al., J Histochem Cytochem 1986, 34 (5):679-682; Conti et al., J Histochem Cytochem 1988, 36 (5):547-550; Ikeda et al. J Histochem Cytochem 1998, 46(3):397-403). It is extremely difficult to extract macromolecules from FFPE clinical specimens due to cross-linking between proteins and nucleic acids. Drs. Clark and Damjanov reported in 1986 that keratin proteins could only be extracted from placenta tissues frozen at −30° C., or fixed in Carnoy's solution, but not from formalin-fixed tissues (Clark et al., J Histochem Cytochem 1986, 34 (5):679-682). Proteins from tissues fixed in non-cross-linking fixatives, such as acetone, alcohol, or Carnoy's solution could be readily extracted, analyzed by SDS-PAGE Coomassie blue staining and immunoblotting (Gillespie et al., Am J Pathol 2002, 160(2):449-457; Shibutani et al., Lab Invest. 2000, 80(2):199-208). However, similar extraction from formalin-fixed tissues generated no detectable bands in Coomassie blue-stained gels and very low amounts of highly degraded bands detectable by polyclonal antibody (Conti et al., J Histochem Cytochem 1988, 36 (5):547-550). These observations led investigators to suspect that formalin-fixation may destroy macromolecules and may not preserve tissues as well as other non-cross-linking fixatives. In the early 1990s, several groups reported that DNA and RNA remained well preserved in FFPE and could be extracted for PCR amplification although the mRNA size would be substantially reduced (von Weizsacker et al., Biochem. Biophys. Res. Commun. 1991, 174:176-180; Neubauer et al., Oncogene 1992, 7:1019-1025; Krafft et al., Nucleic Acids Res. 1999, 27(22):4436-43). Only recently have researchers succeeded in developing protein extraction methods for FFPE tissues (Ikeda et al. J Histochem Cytochem 1998, 46(3):397-403; Izawa et al., Oncol Rep. 2002, 9(6):1313-1318; Murphy et al., Am J Clin Pathol. 2001, 116(1):135-42). However, these processes are destructive and require several hours, substantial amounts of tissues, and high salt concentrations in order to achieve satisfactory protein yields for SDS-PAGE analysis.
The present invention, to be presented in the following sections, overcomes these problems by providing a device and a simple, rapid, and non-destructive molecule extraction (NDME) method which not only can extract high quantity of proteins and nucleic acids from frozen or formalin-fixed paraffin-embedded tissue specimens, but also can maintain the integrity of the tissue morphology and antigenicity after the biological molecules are extracted, which are useful for histopathological studies. Over 500 tissue specimens were tested using this device and the NDME method. The NDME device and method have demonstrated at least the following three potential applications: 1) simultaneous proteomic, genomic studies and histological analysis, including H&E, IHC, and ISH for difficulty clinical cases; 2) performance of retrospective studies for various diseases, particularly those that have not been investigated, and 3) identification of relationships between levels of disease-perturbed proteins and response to drug therapy, ultimately allowing clinicians to not only provide a morphologic diagnosis, but to determine which therapy will yield the greatest response.